Factors governing the assembly of cationic phospholipid-DNA complexes

The interaction of DNA with a novel cationic phospholipid transfection reag ent, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), was investigate d by monitoring thermal effects, particle size, vesicle rupture, and lipid mixing. By isothermal titration calorimetry, the heat of interaction betwee n large unilamellar EDOPC vesicles and plasmid DNA was endothermic at both physiological and low ionic strength, although the heat absorbed was slight ly larger at the higher ionic strength. The energetic driving force for DNA -EDOPC association is thus an increase in entropy, presumably due to releas e of counterions and water. The estimated minimum entropy gain per released counterion was 1.4 cal/mole-degrees K (about 0.7 kT), consistent with prev ious theoretical predictions. All experimental approaches revealed signific ant differences in the DNA-lipid particle, depending upon whether complexes were formed by the addition of DNA to lipid or vice versa. When EDOPC vesi cles were titrated with DNA at physiological ionic strength, particle size increased, vesicles ruptured, and membrane lipids became mixed as the amoun t of DNA was added up to a 1.6:1 (+:-) charge ratio. This charge ratio also corresponded to the calorimetric end point. In contrast, when lipid was ad ded to DNA, vesicles remained separate and intact until a charge ratio of 1 :1 (+:-) was exceeded. Under such conditions, the calorimetric end point wa s 3:1 (+:-). Thus it is clear that fundamental differences in DNA-cationic lipid complexes exist, depending upon their mode of formation. A model is p roposed to explain the major differences between these two situations. Sign ificant effects of ionic strength were observed; these are rationalized in terms of the model. The implications of the analysis are that considerable control can be exerted over the structure of the complex by exploiting vect orial preparation methods and manipulating ionic strength.